RING Finger E3 Ubiquitin Ligase Research Articles

MARCH5 promotes aerobic glycolysis to facilitate ovarian cancer progression via ubiquitinating MPC1.

MARCH5 is a ring-finger E3 ubiquitin ligase located in the outer membrane of mitochondria. A previous study has reported that MARCH5 was up-regulated and contributed to the migration and invasion of OC cells by serving as a competing endogenous RNA. However, as a mitochondrial localized E3 ubiquitin ligase, the function of MARCH5 in mitochondrial-associated metabolism reprogramming in human cancers remains largely unexplored, including OC. We first assessed the glycolysis effect of MARCH5 in OC both in vitro and in vivo. Then we analyzed the effect of MARCH5 knockdown or overexpression on respiratory activity by evaluating oxygen consumption rate, activities of OXPHOS complexes and production of ATP in OC cells with MARCH5. Co-immunoprecipitation, western-blot, and in vitro and vivo experiments were performed to investigate the molecular mechanisms underlying MARCH5-enhanced aerobic glycolysis s in OC. In this study, we demonstrate that the abnormal upregulation of MARCH5 is accompanied by significantly increased aerobic glycolysis in OC. Mechanistically, MARCH5 promotes aerobic glycolysis via ubiquitinating and degrading mitochondrial pyruvate carrier 1 (MPC1), which mediates the transport of cytosolic pyruvate into mitochondria by localizing on mitochondria outer membrane. In line with this, MPC1 expression is significantly decreased and its downregulation is closely correlated with unfavorable survival. Furthermore, in vitro and in vivo assays revealed that MARCH5 upregulation-enhanced aerobic glycolysis played a critical role in the proliferation and metastasis of OC cells. Taken together, we identify a MARCH5-regulated aerobic glycolysis mechanism by degradation of MPC1, and provide a rationale for therapeutic targeting of aerobic glycolysis via MARCH5-MPC1 axis inhibition.

Abstract 1992: A novel RB1 E3 ligase TRIM37 contributes to palbociclib resistance in breast cancer

Abstract The tripartite motif-containing 37 (TRIM37) is a RING finger E3 ubiquitin ligase and is a poor prognostic factor in breast cancer patients. The gene is located in chromosome 17q23, a region amplified in up to 40% of breast cancers. We observed that activation of androgen receptor (AR) could induce resistance to the CDK4/6 inhibitor Palbociclib, the mechanism yet to be clarified. ChIP-sequencing data suggested that AR binds to the TRIM37 promoter, with AR activation resulting in TRIM37 expression. We hypothesized that TRIM37 might be involved in Palbociclib resistance. Using an ER+ve cell line MCF7 and a TNBC cell line MDA-MB 453, we found that overexpression of TRIM37 could induce Palbociclib resistance. TRIM37 overexpression resulted in RB1 protein downregulation, which is the primary downstream target of CDK4/6, while TRIM37 knockdown enhanced RB1 protein expression. Ubiquitination assay demonstrated TRIM37 overexpression increased the level of RB1 protein ubiquitination. The co-immunoprecipitation (Co-IP) assay confirmed that TRIM37 protein could interact with the RB1 protein. These results suggest that TRIM37 could be the E3 ligase that regulates RB1 degradation. Various deletion mutants of TRIM37 were cloned for Co-IP experiments between these mutants and RB1. Results show that the MATH and c-terminal domains of TRIM37 was the essential region for the interaction. Overexpression of the mutant with the TRIM37 MATH and c-terminal domains (TRIM37-MATH/C) could enhance RB1 protein level. This might be due to competition of TRIM37-MATH/C with wild-type TRIM37 for RB1 binding. Our findings suggest TRIM37 is a novel RB1 E3 ligase, and it binds to the RB1 protein via the MATH and C-terminal domains to regulate protein degradation. Our study also suggests that high expression of TRIM37 mediated by AR activation could deplete RB1, which is the target of Palbociclib, eventually leading to resistance. These findings are supportive of the contribution of AR activation to Palbociclib resistance. Citation Format: Chanping You, Ho Tsoi, Pui Sum Man, Larry Ka Yue Chow, Wei Dai, Ui Soon Khoo. A novel RB1 E3 ligase TRIM37 contributes to palbociclib resistance in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1992.

The role of RNF138 in DNA end resection is regulated by ubiquitylation and CDK phosphorylation

Double-strand breaks (DSBs) are DNA lesions that pose a significant threat to genomic stability. The repair of DSBs by the homologous recombination (HR) pathway is preceded by DNA end resection, the 5' to 3' nucleolytic degradation of DNA away from the DSB. We and others previously identified a role for RNF138, a really interesting new gene finger E3 ubiquitin ligase, in stimulating DNA end resection and HR. Yet, little is known about how RNF138's function is regulated in the context of DSB repair. Here, we show that RNF138 is phosphorylated at residue T27 by cyclin-dependent kinase (CDK) activity during the S and G2 phases of the cell cycle. We also observe that RNF138 is ubiquitylated constitutively, with ubiquitylation occurring in part on residue K158 and rising during the S/G2 phases. Interestingly, RNF138 ubiquitylation decreases upon genotoxic stress. By mutating RNF138 at residues T27, K158, and the previously identified S124 ataxia telangiectasia mutated phosphorylation site (Han etal., 2016, ref. 22), we find that post-translational modifications at all three positions mediate DSB repair. Cells expressing the T27A, K158R, and S124A variants of RNF138 are impaired in DNA end resection, HR activity, and are more sensitive to ionizing radiation compared to those expressing wildtype RNF138. Our findings shed more light on how RNF138 activity is controlled by the cell during HR.

Discovery, Optimization, and Biological Evaluation of Arylpyridones as Cbl-b Inhibitors.

Casitas B-lymphoma proto-oncogene-b (Cbl-b), a member of the Cbl family of RING finger E3 ubiquitin ligases, has been demonstrated to play a central role in regulating effector T-cell function. Multiple studies using gene-targeting approaches have provided direct evidence that Cbl-b negatively regulates T, B, and NK cell activation via a ubiquitin-mediated protein modulation. Thus, inhibition of Cbl-b ligase activity can lead to immune activation and has therapeutic potential in immuno-oncology. Herein, we describe the discovery and optimization of an arylpyridone series as Cbl-b inhibitors by structure-based drug discovery to afford compound 31. This compound binds to Cbl-b with an IC50 value of 30 nM and induces IL-2 production in T-cells with an EC50 value of 230 nM. Compound 31 also shows robust intracellular target engagement demonstrated through inhibition of Cbl-b autoubiquitination, inhibition of ubiquitin transfer to ZAP70, and the cellular modulation of phosphorylation of a downstream signal within the TCR axis.

RNF115/BCA2 deficiency alleviated acute liver injury in mice by promoting autophagy and inhibiting inflammatory response

The E3 ubiquitin ligase RING finger protein 115 (RNF115), also known as breast cancer-associated gene 2 (BCA2), has been linked with the growth of some cancers and immune regulation, which is negatively correlated with prognosis. Here, it is demonstrated that the RNF115 deletion can protect mice from acute liver injury (ALI) induced by the treatment of lipopolysaccharide (LPS)/D-galactosamine (D-GalN), as evidenced by decreased levels of alanine aminotransaminase, aspartate transaminase, inflammatory cytokines (e.g., tumor necrosis factor α and interleukin-6), chemokines (e.g., MCP1/CCL2) and inflammatory cell (e.g., monocytes and neutrophils) infiltration. Moreover, it was found that the autophagy activity in Rnf115−/− livers was increased, which resulted in the removal of damaged mitochondria and hepatocyte apoptosis. However, the administration of adeno-associated virus Rnf115 or autophagy inhibitor 3-MA impaired autophagy and aggravated liver injury in Rnf115−/− mice with ALI. Further experiments proved that RNF115 interacts with LC3B, downregulates LC3B protein levels and cell autophagy. Additionally, Rnf115 deletion inhibited M1 type macrophage activation via NF-κB and Jnk signaling pathways. Elimination of macrophages narrowed the difference in liver damage between Rnf115+/+ and Rnf115−/− mice, indicating that macrophages were linked in the ALI induced by LPS/D-GalN. Collectively, for the first time, we have proved that Rnf115 inactivation ameliorated LPS/D-GalN-induced ALI in mice by promoting autophagy and attenuating inflammatory responses. This study provides new evidence for the involvement of autophagy mechanisms in the protection against acute liver injury.

Molecular Landscape of c-CBL Mutation in Adults' Myeloid Malignancies

Background: CBL proteins are a family of RING finger (RF) E3 ubiquitin ligases that regulate proliferative signals via ubiquitination and degradation of tyrosine-phosphorylated signaling protein ( e.g., FLT3, JAK2, MPL). Thus, the loss of CBL activity due to hypomorphic or loss of function (LOF) mutations functionally may result in oncogenic effects. However, simple LOF does not explain the high rates of c-CBL (most abundant CBL isoform, located on chromosome 11)missensemutations affecting a canonical position in the RF domain, catalytic region responsible for ubiquitin ligase activity. We hypothesized that studying the molecular and clinical associations of c-CBL mutations will help clarifying the consequences of its mutations and conceptualize innovative treatment strategies. Methods: We analyzed molecular and clinical features of a cohort of 11036 patients with myeloid neoplasia (MN; 63% AML, 29% MDS, 5% MPN/MDS, 3% MPN) from The Cleveland Clinic Foundation and public series. Clonal burden of c-CBL was analyzed to infer clonal hierarchy and to establish founder vs secondary mutations based on our previously described criteria. Results: Overall, c-CBL mutations were detected in 2.4% (270/11036) of the patients. The most common c-CBL mutations were missense (80%) and mainly occurred in the RF domain and linker region (83% of mutations, Fig1). c-CBL mutations were found in 2% of both primary AML (pAML; 3% of CBF) and sAML/HR-MDS and 8% of CMML. Among CMML patients, mutations were associated with low-risk features (7% of CMML2 vs. 13% of CMML1). The most frequent co-associated mutations were TET2 (22%) and RUNX1 (19%) in all AML, ASXL1 (50%), SRSF2 (25%) and TET2 (25%) in MDS, and ASXL1 (50%), SRSF2 (44%) and JAK2 (28%) in MPN and MPN/MDS. In terms of associations with tyrosine kinase receptors ( FLT3, c-KIT, CSF3R, c-MPL), we found that 18% of c-CBL mutants carried at least one mutation in one the four receptors, contrarily to what expected from previously reported results of c- CBL/FLT3 mutational exclusivity. In most of the cases, c-CBL mutations were subclonal (53%), arguing against its primary driver/initiator function. Indeed, we observed c-CBL mutations as the only lesion in 7% of the cases with no specific disease association (66%, AML; 25%, LR-MDS; 8%, CMML). There was no difference in MN phenotypes between carriers of canonical vs non canonical c-CBL mutations; however, we found that non canonical mutations were more often ancestral or the only founder lesion detected compared to canonical mutations (46 vs 31%, P = 0.031). Del(11q) or monosomy 11 were found in 8 patients, without association with c- CBL VAF >50%. Furthermore, c-CBL VAF>50% was not associated with marrow blast infiltrate, WBC or shorter survival. We then analyzed the effect of c-CBL in cooperation with RAS genes ( NRAS, KRAS, PTPN11, NF1) and found that, in AML, the effect of c-CBL with 2 RAS genes (5 patients, 3%) showed some trends toward higher blast count ( P = 0.079), higher WBC ( P = 0.076) and lower rates of normal cytogenetics compared to the effect of the remaining c-CBL mutant (0 vs 48%, P = 0.061). Conclusions: In sum, our study of the molecular landscape of c-CBL mutations highlights that these lesions occur mainly during disease development with majority of the cases carrying subclonal hits with a weaker effect on disease phenotype and prognosis. Such mutations can be present in both primary and secondary AML (in contrast with previous reports) and additive effects can be observed when c- CBL mutationsco-occur with more than one RAS gene in these disease groups. In some cases, especially in non-canonical mutations, c- CBL could also act as ancestral event and as an independent leukemic driver.

Ring Finger Protein 34 Facilitates Nervous Necrosis Virus Evasion of Antiviral Innate Immunity by Targeting TBK1 and IRF3 for Ubiquitination and Degradation in Teleost Fish.

Ubiquitination, as one of the most prevalent posttranslational modifications of proteins, enables a tight control of host immune responses. Many viruses hijack the host ubiquitin system to regulate host antiviral responses for their survival. Here, we found that the fish pathogen nervous necrosis virus (NNV) recruited Lateolabrax japonicus E3 ubiquitin ligase ring finger protein 34 (LjRNF34) to inhibit the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via ubiquitinating Lateolabrax japonicus TANK-binding kinase 1 (LjTBK1) and interferon regulatory factor 3 (LjIRF3). Ectopic expression of LjRNF34 greatly enhanced NNV replication and prevented IFN production, while deficiency of LjRNF34 led to the opposite effect. Furthermore, LjRNF34 targeted LjTBK1 and LjIRF3 via its RING domain. Of note, the interactions between LjRNF34 and LjTBK1 or LjIRF3 were conserved in different cellular models derived from fish. Mechanically, LjRNF34 promoted K27- and K48-linked ubiquitination and degradation of LjTBK1 and LjIRF3, which in turn diminished LjTBK1-induced translocation of LjIRF3 from the cytoplasm to the nucleus. Ultimately, NNV capsid protein (CP) was found to bind with LjRNF34, CP induced LjTBK1 and LjIRF3 degradation, and IFN suppression depended on LjRNF34. Our finding demonstrates a novel mechanism by which NNV CP evaded host innate immunity via LjRNF34 and provides a potential drug target for the control of NNV infection. IMPORTANCE Ubiquitination plays an essential role in the regulation of innate immune responses to pathogens. NNV, a type of RNA virus, is the causal agent of a highly destructive disease in a variety of marine and freshwater fish. A previous study reported NNV could hijack the ubiquitin system to manipulate the host's immune responses; however, how NNV utilizes ubiquitination to facilitate its own replication is not well understood. Here, we identified a novel distinct role of E3 ubiquitin ligase LjRNF34 as an IFN antagonist to promote NNV infection. NNV capsid protein utilized LjRNF34 to target LjTBK1 and LjIRF3 for K27- and K48-linked ubiquitination and degradation. Importantly, the interactions between LjRNF34 and CP, LjTBK1, or LjIRF3 are conserved in different cellular models derived from fish, suggesting it is a general immune evasion strategy exploited by NNV to target the IFN response via RNF34.

RING finger E3 ubiquitin ligase gene TaAIRP2-1B controls spike length in wheat.

E3 ubiquitin ligase genes play important roles in the regulation of plant development. They have been well studied in plants, but have not been sufficiently investigated in wheat. Here, we identified a highly expressed RING finger E3 ubiquitin ligase gene TaAIRP2-1B (ABA-insensitive RING protein 2) in wheat spike. Sequence polymorphism and association analysis showed that TaAIRP2-1B is significantly associated with spike length under various conditions. The genotype with haplotype Hap-1B-1 of TaAIRP2-1B has a longer spike than that of Hap-1B-2, and was positively selected in the process of wheat breeding in China. Moreover, the TaAIRP2-1B-overexpressing rice lines have longer panicles compared with wild-type plants. The expression levels of TaAIRP2-1B in Hap-1B-1 accessions were higher than in Hap-1B-2 accessions. Further study revealed that the expression of TaAIRP2-1B was negatively regulated by TaERF3 (ethylene-responsive factor 3) via binding to the Hap-1B-2 promoter, but not via binding of Hap-1B-1. Additionally, several candidate genes interacting with TaAIRP2-1B were obtained by screening the cDNA library of wheat in yeast cells. It was found that TaAIRP2-1B interacted with TaHIPP3 (heavy metal-associated isoprenylated protein 3) and promoted TaHIPP3 degradation. Our study demonstrates that TaAIRP2-1B controls spike length, and the haplotype Hap-1B-1 of TaAIRP2-1B is a favorable natural variation for spike length enhancement in wheat. This work also provides genetic resources and functional markers for wheat molecular breeding.

Sorafenib regulates c-CBL gene-mediated chemoresistance in acute myeloid leukemia cells

ABSTRACTChemotherapeutic regimens containing sorafenib are widely used in salvage treatment for patients with relapsed and refractory acute leukemia, especially those with FLT3-ITD mutations. However, the therapeutic effects in individuals are heterogeneous, and the effective maintenance period is relatively short. Our clinical analysis showed patients with high c-kit (CD117) expression in leukemia cells generally had a better response to sorafenib, but the reason for this finding was not clear. c-kit (CD117) is a receptor tyrosine kinase, and its signal inactivation and hydrolytic metabolism are regulated by the CBL protein, a Ring finger E3 ubiquitin ligase, encoded by the c-CBL gene. And we also found that the c-CBL gene expression in refractory and relapsed patients was significantly lower than that in healthy hematopoietic stem cell donors. Therefore, we assumed that there is a relationship among c-CBL gene function, high expression of c-kit (CD117) and a better clinical response to sorafenib. To confirm this hypothesis, we packaged interfering lentiviruses and overexpressed adenoviruses targeting the c-CBL gene respectively, and infected leukemia cell lines with these viruses to regulate the expression of the c-CBL gene, and observed the subsequent changes of these cells in various biological behaviors. Our results showed when the c-CBL gene was silenced, the cells proliferation was accelerated, drug sensitivity to cytarabine or sorafenib was decreased, and apoptosis ratio was decreased. And all these phenomena were reversed when the gene was overexpressed, which confirmed the expression of c-CBL gene was related to drug resistance in leukemia cells. At last, we explored the possible molecular mechanisms underlying these phenomena.

The D14-SDEL1-SPX4 cascade integrates the strigolactone and phosphate signalling networks in rice.

Modern agriculture needs large quantities of phosphate (Pi) fertilisers to obtain high yields. Information on how plants sense and adapt to Pi is required to enhance phosphorus-use efficiency (PUE) and thereby promote agricultural sustainability. Here, we show that strigolactones (SLs) regulate riceroot developmental and metabolic adaptations to low Pi, by promoting efficient Pi uptake and translocation from roots to shoots. Low Pi stress triggers the synthesis of SLs, which dissociate the Pi central signalling module of SPX domain-containing protein (SPX4) and PHOSPHATE STARVATION RESPONSE protein (PHR2), leading to the release of PHR2 into the nucleus and activating the expression of Pi-starvation-induced genes including Pi transporters. The SL synthetic analogue GR24 enhances the interaction between the SL receptor DWARF 14 (D14) and a RING-finger ubiquitin E3 ligase (SDEL1). The sdel mutants have a reduced response to Pi starvation relative to wild-type plants, leading to insensitive root adaptation to Pi. Also, SLs induce the degradation of SPX4 via forming the D14-SDEL1-SPX4 complex. Our findings reveal a novel mechanism underlying crosstalk between the SL and Pi signalling networks in response to Pi fluctuations, which will enable breeding of high-PUE crop plants.

ZNRF2 as an oncogene is transcriptionally regulated by CREB1 in breast cancer models.

E3 ubiquitin ligase Zinc and Ring Finger 2 (ZNRF2) has been demonstrated to be engaged in the development of multiple cancers. Nevertheless, the function of ZNRF2 in breast cancer (BC) still unclear. In this work, we firstly analyzed the differentially expressed genes in BC by bioinformatics and found that ZNRF2 was highly expressed in BC. Consistently, we further confirmed that ZNRF2 was upregulated in BC tissues compared with adjacent normal tissues, and this was positively correlated with the poor prognosis and the higher pathological grades of patients with BC. Functional assays performed on HCC1937 and MCF-7 cells indicated that silencing of ZNRF2 suppressed cell proliferation, as evidenced by the decrease in the expression of cyclin A, PCNA and cyclin D1. Flow cytometry and Hoechst staining showed that knockdown of ZNRF2 induced cell apoptosis, which was verified by the upregulation of apoptosis genes such as Bax, cleaved PARP and Bim. ZNRF2 knockdown also inhibited in vivo tumor growth. But, instead, ZNRF2-overexpressed BC cells exhibited obvious malignant phenotypes. Additionally, we observed that cAMP response element binding protein 1 (CREB1) directly bound to the promoter sequence of ZNRF2 and thus activating its transcription, suggesting that ZNRF2 is transcriptionally regulated by CREB1. Additionally, ZNRF2 knockdown could reverse the proliferation-promoting action of CREB1 on BC cells, Hence, this study demonstrated that ZNRF2 might serve as a prospective therapeutic target for BC.

RNF125 attenuates hepatocellular carcinoma progression by downregulating SRSF1-ERK pathway.

Hepatocellular carcinoma (HCC) is one of the most deadly malignant cancers worldwide. Research into the crucial genes responsible for maintaining the aggressive behaviour of cancer cells is important for the clinical treatment of HCC. The purpose of this study was to determine whether the E3 ubiquitin ligase Ring Finger Protein 125 (RNF125) plays a role in the proliferation and metastasis of HCC. RNF125 expression in human HCC samples and cell lines was investigated using TCGA dataset mining, qRT‒PCR, western blot, and immunohistochemistry assays. In addition, 80 patients with HCC were studied for the clinical value of RNF125. Furthermore, the molecular mechanism by which RNF125 contributes to hepatocellular carcinoma progression was determined with mass spectrometry (MS), coimmunoprecipitation (Co-IP), dual-luciferase reporter assays, and ubiquitin ladder assays. We found that RNF125 was markedly downregulated in HCC tumour tissues, which was associated with a poor prognosis for patients with HCC. Moreover, the overexpression of RNF125 inhibited HCC proliferation and metastasis both in vitro and in vivo, whereas the knockdown of RNF125 exerted antithetical effects. Mechanistically, mass spectrometry analysis revealed a protein interaction between RNF125 and SRSF1, and RNF125 accelerated the proteasome-mediated degradation of SRSF1, which impeded HCC progression by inhibiting the ERK signalling pathway. Furthermore, RNF125 was detected to be the downstream target of miR-103a-3p. In this study, we identified that RNF125 is a tumour suppressor in HCC and inhibits HCC progression by inhibiting the SRSF1/ERK pathway. These findings provide a promising treatment target for HCC.

RING finger gene 180 inhibits osteosarcoma progression through regulating chromobox homolog 4 ubiquitination

ABSTRACT Osteosarcoma (OS) is still the most common malignant bone tumor whose etiology remains largely unclear. Here, we aimed to investigate the role of a novel E3 ubiquitin ligase RING finger gene 180 (RNF180) in OS progression. RNF180 was significantly down-regulated in both OS tissues and cell lines. We up-regulated RNF180 using over-expression vector and knocked down RNF180 using specific short hairpin RNAs in OS cell lines. RNF180 over-expression inhibited the viability and proliferation yet promoted apoptosis in OS cells, while RNF180 knockdown showed the opposite effects. RNF180 also suppressed tumor growth and lung metastasis in mouse model, accompanied with elevated E-cadherin level and decreased ki-67 level. Besides, chromobox homolog 4 (CBX4) was predicted as a substrate of RNF180. RNF180 and CBX4 were both localized mainly in nucleus and their interaction was validated. RNF180 aggravated the decline of CBX4 level after cycloheximide treatment. RNF180 also promoted the ubiquitination of CBX4 in OS cells. Furthermore, CBX4 was significantly up-regulated in OS tissues. RNF180 also up-regulated Kruppel like factor 6 (KLF6) yet down-regulated RUNX family transcription factor 2 (Runx2) in OS, which served as downstream targets of CBX4. In addition, RNF180 inhibited migration, invasion and epithelial-mesenchymal transition (EMT) in OS cells, which were partially abolished by CBX4 over-expression. In conclusion, our findings demonstrated that RNF180 inhibits OS development via regulating CBX4 ubiquitination, and RNF180-CBX4 axis is a potential therapeutic target for OS treatment.

Phosphorylation and ubiquitination of OsWRKY31 are integral to OsMKK10-2-mediated defense responses in rice.

Mitogen-activated protein kinase (MPK) cascades play vital roles in plant innate immunity, growth, and development. Here, we report that the rice (Oryza sativa) transcription factor gene OsWRKY31 is a key component in a MPK signaling pathway involved in plant disease resistance in rice. We found that the activation of OsMKK10-2 enhances resistance against the rice blast pathogen Magnaporthe oryzae and suppresses growth through an increase in jasmonic acid and salicylic acid accumulation and a decrease of indole-3-acetic acid levels. Knockout of OsWRKY31 compromises the defense responses mediated by OsMKK10-2. OsMKK10-2 and OsWRKY31 physically interact, and OsWRKY31 is phosphorylated by OsMPK3, OsMPK4, and OsMPK6. Phosphomimetic OsWRKY31 has elevated DNA-binding activity and confers enhanced resistance to M. oryzae. In addition, OsWRKY31 stability is regulated by phosphorylation and ubiquitination via RING-finger E3 ubiquitin ligases interacting with WRKY 1 (OsREIW1). Taken together, our findings indicate that modification of OsWRKY31 by phosphorylation and ubiquitination functions in the OsMKK10-2-mediated defense signaling pathway.

Degradation of Ubiquitin-Editing Enzyme A20 following Autophagy Activation Promotes RNF168 Nuclear Translocation and NF-κB Activation in Lupus Nephritis

The correlation between ubiquitin-editing enzyme A20 and E3 ubiquitin ligase ring finger protein (RNF) 168 has been reported to be critical for repair of DNA damage. This study aimed to evaluate the potential role of this regulatory interaction in the pathogenesis of lupus nephritis (LN). The expression of RNF168 and A20 was measured in the podocytes derived from MRL/lpr murine lupus as well as patients with LN. Cell-based studies using renal podocytes bearing silenced RNF168, over-expressed A20, autophagy-related gene (Atg) 5 (a ubiquitin-like modifier), or silenced Atg5 were used to assess the effect of RNF168, A20, and Atg5 on DNA damage repair and nuclear factor kappa-B (NF-κB) activation in LN. It was found that podocyte autophagy was over-activated in LN and the abnormal podocyte autophagy led to down-regulation of A20, up-regulation of RNF168, and activation of the NF-κB. RNF168 silencing or A20 restoration inhibited activation of NF-κB pathway and promoted repair of DNA damage, where the level of autophagy was not changed. Activated A20 in podocytes weakened the promoting action of cell autophagy on RNF168. The current results suggest that RNF168 dysfunction may be involved in the pathogenesis of LN via down-regulation of A20 expression. Autophagy and RNF168 may be therapeutic targets for the prevention and treatment of LN.

RNF126-Mediated MRE11 Ubiquitination Activates the DNA Damage Response and Confers Resistance of Triple-Negative Breast Cancer to Radiotherapy.

Triple-negative breast cancer (TNBC) has higher molecular heterogeneity and metastatic potential and the poorest prognosis. Because of limited therapeutics against TNBC, irradiation (IR) therapy is still a common treatment option for patients with lymph nodes or brain metastasis. Thus, it is urgent to develop strategies to enhance the sensitivity of TNBC tumors to low-dose IR. Here, the authors report that E3 ubiquitin ligase Ring finger protein 126 (RNF126) is important for IR-induced ATR-CHK1 pathway activation to enhance DNA damage repair (DDR). Mechanistically, RNF126 physically associates with the MRE11-RAD50-NBS1 (MRN) complex and ubiquitinates MRE11 at K339 and K480 to increase its DNA exonuclease activity, subsequent RPA binding, and ATR phosphorylation, promoting sustained DDR in a homologous recombination repair-prone manner. Accordingly, depletion of RNF126 leads to increased genomic instability and radiation sensitivity in both TNBC cells and mice. Furthermore, it is found that RNF126 expression is induced by IR activating the HER2-AKT-NF-κB pathway and targeting RNF126 expression with dihydroartemisinin significantly improves the sensitivity of TNBC tumors in the brain to IR treatment in vivo. Together, these results reveal that RNF126-mediated MRE11 ubiquitination is a critical regulator of the DDR, which provides a promising target for improving the sensitivity of TNBC to radiotherapy.

Ring finger protein 126 promotes breast cancer metastasis and serves as a potential target to improve the therapeutic sensitivity of ATR inhibitors

Background/aimsThis study explores the relationship between the E3 ubiquitin ligase Ring finger protein 126 (RNF126) and early breast cancer metastasis and tests the hypothesis that RNF126 determines the efficacy of inhibitors targeting Ataxia telangiectasia mutated and Rad3-related kinase (ATR).MethodsVarious metastasis-related genes were identified by univariable Cox proportional hazards regression analysis based on the GSE11121 dataset. The RNF126-related network modules were identified by WGCNA, whereas cell viability, invasion, and migration assays were performed to evaluate the biological characteristics of breast cancer cells with or without RNF126 knockdown. MTT, immunoblotting, immunofluorescence, and DNA fiber assays were conducted to determine the efficiency of ATR inhibitor in cells with or without RNF126 knockdown.ResultsRNF126 was associated with early breast cancer metastasis. RNF126 promoted breast cancer cell proliferation, growth, migration, and invasion. ATR inhibitors were more effective at killing breast cancer cells with intact RNF126 due to replication stress compared with the corresponding cells with RNF126 knockdown. Cyclin-dependent kinase 2 (CDK2) was involved in regulating replication stress in breast cancer cells with intact RNF126.ConclusionA high level of expression of RNF126 in early breast cancer patients without lymph node metastases may indicate a high-risk type of metastatic disease, possibly due to RNF126, which may increase breast cancer cell proliferation and invasion. RNF126-expressing breast cancer cells exhibit CDK2-mediated replication stress that makes them potential targets for ATR inhibitors.

RNF43 induces the turnover of protease-activated receptor 2 in colon cancer.

Post-translational modification of G-protein coupled receptors (GPCRs) plays a central role in tissue hemostasis and cancer. The molecular mechanism of post-translational regulation of protease-activated receptors (PARs), a subgroup of GPCRs is yet understudied. Here we show that the cell-surface transmembrane E3 ubiquitin ligase ring finger 43 (RNF43) is a negative feedback regulator of PAR2 , impacting PAR2 -induced signaling and colon cancer growth. RNF43 co-associates with PAR2 , promoting its membrane elimination and degradation as shown by reduced cell surface biotinylated PAR2 levels and polyubiquitination. PAR2 degradation is rescued by R-spondin2 in the presence of leucine-rich repeat-containing G-protein-coupled receptor5 (LGR5). In fact, PAR2 acts jointly with LGR5, as recapitulated by increased β-catenin levels, transcriptional activity, phospho-LRP6, and anchorage-independent colony growth in agar. Animal models of the chemically induced AOM/DSS colon cancer of wt versus Par2/f2rl1 KO mice as also the 'spleen-liver' colon cancer metastasis, allocated a central role for PAR2 in colon cancer growth and development. RNF43 is abundantly expressed in the Par2/f2rl1 KO-treated AOM/DSS colon tissues while its level is very low to nearly null in colon cancer adenocarcinomas of the wt mice. The same result is obtained in the 'spleen-liver' model of spleen-inoculated cells, metastasized to the liver. High RNF43 expression is observed in the liver upon shRNA -Par2 silencing. "Limited-dilution-assay" performed in mice in-vivo, assigned PAR2 as a member of the cancer stem cell niche compartment. Collectively, we elucidate an original regulation of PAR2 oncogene, a member of cancer stem cells, by RNF43 ubiquitin ligase. It impacts β-catenin signaling and colon cancer growth.

E3 Ubiquitin Ligase RNF125 Suppresses Immune Escape in Head and Neck Squamous Cell Carcinoma by Regulating PD-L1 Expression.

Head and neck squamous cell carcinoma (HNSCC) is a lethal malignancy. Given the essential roles of E3 ligases in cancer immunotherapies, this paper explored the effect of E3 ubiquitin ligase ring finger protein 125 (RNF125) on immune escape in HNSCC. After delivering overexpressed (oe)-RNF125, interferon-gamma, or oe-programmed death-ligand 1 (PD-L1) into HNSCC cells and cell culture with carboxyfluorescein succinimidyl ester-labeled CD8+ T cells, RNF125 and PD-L1 levels were determined via RT-qPCR and Western blot, with HNSCC cell behaviors assessed via colony formation assay, Transwell assays and flow cytometry, and inflammatory factors measured via ELISA. PD-L1 ubiquitination level and PD-L1's interaction with RNF125 were analyzed via co-immunoprecipitation. The in vivo action of RNF125 on HNSCC was validated via nude mouse tumorigenicity assay. Briefly, RNF125 was weakly expressed in HNSCC cells. RNF125 overexpression inhibited immune escape of HNSCC cells, evidenced by decreased TSCCA cell proliferation, migration, and invasion, increased CD8+ T cell proliferation, and elevated IL-2 and TNF-α levels. RNF125 downregulated PD-L1 in TSCCA cells and facilitated PD-L1 ubiquitinational degradation. PD-L1 overexpression partially abated RNF125-mediated suppression on TSCCA cell immune escape. Moreover, RNF125 suppressed tumorigenesis and tumor growth in vivo. Overall, RNF125 promoted PD-L1 ubiquitinational degradation, hence inhibiting immune escape in HNSCC.

Tumor elimination by clustered microRNAs miR-306 and miR-79 via noncanonical activation of JNK signaling.

JNK signaling plays a critical role in both tumor promotion and tumor suppression. Here, we identified clustered microRNAs (miRNAs) miR-306 and miR-79 as novel tumor-suppressor miRNAs that specifically eliminate JNK-activated tumors in <i>Drosophila</i>. While showing only a slight effect on normal tissue growth, miR-306 and miR-79 strongly suppressed growth of multiple tumor models, including malignant tumors caused by Ras activation and cell polarity defects. Mechanistically, these miRNAs commonly target the mRNA of an E3 ubiquitin ligase ring finger protein 146 (RNF146). We found that RNF146 promotes degradation of tankyrase (Tnks), an ADP-ribose polymerase that promotes JNK activation in a noncanonical manner. Thus, downregulation of RNF146 by miR-306 and miR-79 leads to hyper-enhancement of JNK activation. Our data show that, while JNK activity is essential for tumor growth, elevation of miR-306 or miR-79 overactivate JNK signaling to the lethal level via noncanonical JNK pathway and thus eliminate tumors, providing a new miRNA-based strategy against cancer.